Fluorescein dyes, commonly used for identifying oxidative stress in bacteria, are likely ineffective, according to researchers in the US. This research follows a 2024 study by the same authors that concluded another popular method for detecting oxidative stress was also unproductive. Together, the two papers imply that the notion of reactive oxygen species influencing the mechanism of bactericidal antibiotics is likely faulty, although this view has sparked debate among other scientists. Comprehending how antibiotics function is essential, as understanding their action aids in the development of new medications.
Certain antibiotic agents, like doxorubicin, now primarily known as an anticancer treatment, exert cytotoxic effects by producing reactive oxygen species including superoxide, hydrogen peroxide, and hydroxyl radicals. In 2007, a group led by James Collins – formerly at Boston University in the US – suggested that all antibiotics, including widely used antibacterial agents like penicillin, eliminated bacteria through the action of reactive oxygen species.
Intense debate arose in 2013 with the release of two studies contesting this claim. One by James Imlay at the University of Illinois, Urbana–Champaign and colleagues sought but failed to find additional evidence of oxidative stress in Escherichia coli, such as the activation of a transcription factor that responds to hydrogen peroxide. Nonetheless, the hypothesis retains considerable support, with researchers proposing that reactive oxygen species facilitate various bactericidal activities from nanoparticles, solar radiation, and other sources.
The hypothesis initially relied on two primary pieces of evidence: firstly, that bacteria supplemented with antioxidants displayed greater resistance to antibiotics. Secondly, dyes – typically derivatives of dichlorofluorescein diacetate – that fluoresce upon oxidation appeared to brighten significantly in cells exposed to antibiotics, ostensibly indicating oxidative stress. ‘It would have appeared that in those initial papers, those two distinct approaches corroborated one another, so in later studies, researchers might have only utilized one of them,’ states Imlay.
In 2024, Imlay and his colleague Sergey Korshunov published findings indicating that the first method lacked reliability. They demonstrated that administering antioxidants to cells generally inhibited their growth, thereby making slower-growing cells inherently more resilient to stress. In their latest work, they focused on the dyes.
Imlay and Korshunov present two primary arguments that challenge the efficacy of these dyes as indicators of oxidative stress. First, they demonstrated that bacteria in an oxygen-depleted environment produced the same amount of fluorescence as those in a high-oxygen setting, indicating that the anoxic bacteria were oxidizing the dye through other agents like flavins and quinones. Secondly, they discovered that bacteria expelled the dyes. ‘Bacteria possess numerous efflux pumps that constantly eject molecules, and it turns out they expel these dyes,’ explains Imlay. ‘Any stress that reduces metabolic activity decreases the cell’s energy charge, causing the pumps to reduce dye expulsion. Thus, when stress is applied to cells [for example, antibiotics], there’s an accumulation of dye that has been oxidized by agents like flavins, and this rise in cellular dye signal was incorrectly interpreted as proof of reactive oxygen species.’ Chemicals known to inhibit efflux pump functionality also replicated this phenomenon.
Imlay asserts that the premise that antibiotics operate via reactive oxygen species should be reconsidered. ‘We’re not asserting [the antibiotics] are ineffective,’ Imlay states. ‘We’re asserting that their mechanism of action does not involve oxidative stress.’ He mentions that all bactericidal antibiotics possess mechanisms that do not rely exclusively on the presence of oxygen, explaining their effectiveness against bacteria in low-oxygen conditions. Future studies on oxidative stress, he suggests, should emphasize markers like enzymes. ‘Our lab is exploring which enzymes are easiest for researchers to measure in terms of activity as reliable indicators of oxidative stress,’ he states.
Several researchers contacted by Chemistry World concurred with Imlay that fluorescein dyes are unreliable indicators of oxidative stress in bacteria, yet some remain confident in the assertion that various antibiotic classes function, in part, by initiating oxidative stress. ‘Over a decade ago, we acknowledged the mounting concerns regarding the dyes typically used to assess reactive oxygen species,’ remarks Collins, now at the Massachusetts Institute of Technology. ‘In response, we conducted a comprehensive study utilizing a broad range of assays to directly quantify oxidative damage to essential cellular components, including proteins, DNA, and RNA.’ Within half an hour of treatment with various antibiotics, E. coli displayed common indicators of oxidative stress, which subsequently diverged into distinct causes of cell death. ‘This research … clearly demonstrated that many bactericidal antibiotics induce oxidative stress as part of their mechanism of action.’